Automated quantification of vomeronasal glomeruli number, size, and color composition after immunofluorescent staining

ABSTRACT Glomeruli are neuropil rich regions of the main or accessory olfactory bulbs where the axons of olfactory or vomeronasal neurons and dendrites of mitral/tufted cells form synaptic connections. In the main olfactory system olfactory sensory neurons (OSNs) expressing the same receptor innervate one or two glomeruli. However, in the accessory olfactory system, vomeronasal sensory neurons (VSNs) expressing the same receptor can innervate up to 30 different glomeruli in the accessory olfactory bulb (AOB). Genetic mutation disrupting genes with a role in defining the identity/diversity of olfactory and vomeronasal neurons can alter number and size of glomeruli. Interestingly, two cell surface molecules, Kirrel2 and Kirrel3, have been indicated to play a critical role in the organization of axons into glomeruli in the AOB. Being able to quantify differences in glomeruli features such as number, size or immunoreactivity for specific markers is an important experimental approach to validate the role of specific genes in controlling neuronal connectivity and circuit formation in control or mutant animals. Since the manual recognition and quantification of glomeruli on digital images is a challenging and time-consuming task, we generated a program in Python able to identify glomeruli in digital images and quantify their properties, such as size, number, and pixel intensity. Validation of our program indicates that our script is a fast and suitable tool for high throughput quantification of glomerular features of mouse lines with different genetic makeup.